Medium-chain length fatty acids, glycerides and analogues as neutrophil survival and activation factors

ABSTRACT

A composition and method for promoting neutrophil survival and activation such as the treatment of neutropenia arising as an undesirable side effect of chemotherapy and radiation therapy. A composition containing medium-chain fatty acids, such as Capri acid or caprylic acid, or salts or triglycerides thereof, or mono- or diglycerides or other analogues thereof or medium-chain triglycerides (MCT) is administered to a human or animal needing treatment in an amount sufficient to reduce or eliminate neutropenia. The composition is administered in an amount effective to treat the disorder. The methods are also useful in the management of bone narrow transplantation and in the treatment of various neutropenic diseases.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation application of application U.S. Ser.No. 10/475,266, filed Mar. 4, 2004 now U.S. Pat. No. 7,745,488; which isa National Stage Application of International Application NumberPCT/CA02/00535, filed Apr. 18, 2002, which claims the benefit of U.S.Provisional Application Ser. No. 60/284,458, filed Apr. 18, 2001, whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the prevention and/or treatment ofneutropenia. This includes the treatment of neutropenia associated withthe use of chemotherapy and radiotherapy as well as treatment ofneutropenia arising from infections, hematologic diseases andnutritional deficiencies. The present invention also relates generallyto reducing drug toxicity and enhancing drug efficacy. In particular,the present invention relates to the use of medium-chain length fattyacids such as capric acid, caprylic acid, or salts or triglyceridesthereof or mono- or diglycerides or other analogues thereof as aneutrophil survival and activation factor or bone marrow stem cellproliferation factor.

BACKGROUND OF THE INVENTION

Chemotherapy refers to the use of cytotoxic agents such as, but notlimited to, cyclophosphamide, doxorubicin, daunorubicin, vinblastine,vincristine, bleomycin, etoposide, topotecan, irinotecan, taxotere,taxol, 5-fluorouracil, methotrexate, gemcitabine, cisplatin, carboplatinor chlorambucil in order to eradicate cancer cells and tumors. However,these agents are non-specific and, particularly at high doses, they aretoxic to normal and rapidly dividing cells. This often leads to variousside effects in patients undergoing chemotherapy and radiation therapy.Myelosuppression, a severe reduction of blood cell production in bonemarrow, is one such side effect. It is characterized by leukopenia,neutropenia and thrombocytopenia. Severe chronic neutropenia(idiopathic, cyclic, and congenital) is also characterized by aselective decrease in the number of circulating neutrophils and anenhanced susceptibility to bacterial infections.

The essence of treating cancer with chemotherapeutic drugs is to combinea mechanism of cytotoxicity with a mechanism of selectivity for highlyproliferating tumor cells over host cells. However, it is rare forchemotherapeutic drugs to have such selectivity. The cytotoxicity ofchemotherapeutic agents limits administrable doses, affects treatmentcycles and seriously jeopardizes the quality of life of the oncologicpatient.

Although other normal tissues may also be adversely affected, bonemarrow is particularly sensitive to proliferation-specific treatmentssuch as chemotherapy or radiation therapy. Acute and chronic bone marrowtoxicity is a common side effect of cancer therapies which leads todecreases in blood cell counts and anemia, leukopenia, neutropenia,agranulocytosis and thrombocytopenia. One cause of such effects is adecrease in the number of hematopoietic cells (e.g., pluripotent stemcells and other progenitor cells) caused by both a lethal effect ofcytotoxic agents or radiation on these cells and by differentiation ofstem cells provoked by a feed-back mechanism induced by the depletion ofmore mature marrow compartments. The second cause is a reduction inself-renewal capacity of stem cells, which is also related to bothdirect (mutation) and indirect (agmg of stem cell population) effects.(Tubiana, M., et al., Radiotherapy and Oncology 29: 1-17, 1993). Thus,cancer treatments often result in a decrease in PolymorphonuclearNeutrophils (PMN) or neutropenia. PMN are the first line of defenseagainst invading pathogens and play a central role during acuteinflammation, their primary function being the phagocytosis and killingof the infectious agents. To accomplish this role, PMN leave thecirculation in response to chemotactie factors and enter in the affectedarea to exert their biological functions. In individuals exhibitingnormal blood cell counts, neutrophils constitute approximately 60% ofthe total leukocytes. (SI Units Conversion Guide, 66-67 (1992), NewEngland Journal of Medicine Books). However, as many as one in threepatients receiving chemotherapy treatment for cancer may suffer fromneutropenia. Mean normal neutrophil counts for healthy human adults areon the order of 4400 cells/μL, with a range of 1800-7700 cells/μL. Acount of 1,000 cells to 500 cells/μL is moderate neutropenia and a countof 500 cells/μL or less is severe neutropenia. Patients inmyelosuppressive states are prone to infection and frequently sufferfrom blood-clotting disorders, requiring hospitalization. Lack ofneutrophils and platelets is the leading cause of morbidity andmortality following cancer treatments and contributes to the high costof cancer therapy. In these above-mentioned conditions, the use of anyagent capable of inhibiting neutrophil apoptosis or stimulatingneutrophil activation and mobilization can be of therapeutic value.Efforts to restore the patient's immune system after chemotherapyinvolves the use of hematopoietic growth factors to stimulate remainingstem cells to proliferate and differentiate into mature infectionfighting cells.

In bone marrow transplantation, a phenomenon known as “mobilization” hasalso been exploited to harvest greater numbers of stem/progenitor cellsfrom peripheral blood. This method is currently used for autologous orallogeneic bone marrow transplantation. Growth factors are used toincrease the number of peripheral progenitor stem cells to be harvestedbefore myeloablative therapy and infusion of progenitor stem cells.

Post-therapy bone marrow transplantation can also counter neutropenia.However, these treatments require 10-15 days of treatment which leavespatients vulnerable to infection. Agents capable of stimulating bonemarrow stem cells can facilitate and accelerate stem cells engraftmentthus shortening the neutropenic window following bone marrowtransplantation.

Although hematopoietic growth factors such as granulocyte-macrophagecolony stimulating factor (GM-CSF) and granulocyte colony stimulatingfactor (G-CSF) can exert such actions, their use is expensive since theyhave to be produced by recombinant technology. Such post-therapeuticameliorative treatments are unnecessary if patients are “chemoprotected”from immune suppression.

Therefore, there is a need for novel compositions and methods to reducethe undesirable side effects of myelosuppressive states induced bychemotherapy and radiation therapy.

SUMMARY OF THE INVENTION

The present invention satisfies the need for chemoprotective agents byproviding a novel method for the stimulation of the hematopoietic systemin a mammal, including a human. The present invention also provides anovel method for treating the myelosuppressive effects of chemotherapyand radiotherapy, and any other situation in which the stimulation ofthe hematopoietic system can be of therapeutic value such as, but notlimited to, bone marrow transplantation and chronic neutropenia, as wellas neutropenia resulting from infections, hematologic diseases andnutritional deficiencies. This method assists the hematopoietic systemin countering myelosuppression, increasing neutrophil survival andactivation, in patients undergoing such treatment.

In accordance with this method, a composition containing one or moremedium-chain length fatty acids such as capric acid, caprylic acid, orsalts or triglycerides thereof or mono- or diglycerides or otheranalogues in a pharmaceutically acceptable carrier is administered to amammal, particularly humans, in an amount effective to prevent or treatneutropenia such as for reducing the adverse effects of chemotherapy andradiation therapy and for treating neutropenia arising from infections,hematologic diseases and nutritional deficiencies.

Accordingly, it is an object of the present invention to providecompositions using capric acid, caprylic acid, lauric acid or metallicsalts (sodium, potassium, calcium, magnesium) thereof, or triglyceridesthereof, or mono- or diglycerides or other analogues thereof for theproduction of chemoprotective pharmaceutical compositions as a singleagent or as a combination of two or more agents with and/or withoutother chemotherapeutic agents or such drugs which induce a state ofmyelosuppression.

Another object of the present invention relates to the use of capricacid, caprylic acid or sodium salts or triglycerides thereof or mono- ordiglycerides thereof or related compounds as a hematopoiesis stimulatingfactor.

Furthermore, the present invention includes compositions containingcapric acid, caprylic acid or sodium salts or triglycerides thereof ormono- or diglycerides or other analogues thereof and the use of suchcompounds for the treatment of myelo suppression and subsequentimmunosuppression.

An object of the invention relates also to the use of capric acid,caprylic acid or sodium salts or triglycerides thereof or mono- ordiglycerides or other analogues thereof for the treatment of patientswith severe chronic neutropenia.

Yet another object of the present invention relates to the use of capricacid, caprylic acid or sodium salts or triglycerides thereof or mono- ordiglycerides or other analogues thereof as a neutrophil survival andactivation factor.

The present invention also relates to the use of capric acid, caprylicacid or sodium salts or triglycerides thereof or mono- or diglyceridesor other analogues thereof in conditions where neutrophil mobilizationcan be of therapeutic value such as autologus or allogeneic bone marrowtransplantation.

It is an object of the present invention to provide a method effectivefor providing chemoprotection of a mammal, including a human.

Another object of the present invention is to provide a method effectivefor increasing the effectiveness of chemotherapy and radiation therapyin a mammal, including a human.

Yet another object of the invention is to provide methods for using moreusual dosages, or even increasing the dose of chemotherapeuticcompositions necessary to achieve a better therapeutic benefit, whileavoiding increased side-effects.

Another object of the present invention is to provide a method effectivefor reducing or eliminating chemotherapy-induced neutropenia in amammal, including a human.

Still another object of the present invention is to provide a method fortreating neutropenia arising from hematologic diseases such as chronicidiopathic neutropenias, cyclic neutropenia, lazy-leukocyte syndrome,Chédiak-Higashi syndrome leukemia and aplastic anemia.

Yet another object of the present invention is to provide a method fortreating neutropenia arising from infections such as viral (for example,HIV, measles, hepatitis, yellow fever, mononucleosis) and bacterial (forexample, typhoid, paratyphoid, brucellosis) infections.

Finally, another object of the present invention is to provide a methodthat causes minimal or no adverse side effects in the recipient.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiment and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of MCT on PMN apoptosis.

FIG. 2 shows the effect of MCT on PMN phagocytosis.

FIGS. 3A and 3B show the effect of doxorubicin on PMN apoptosis.

FIG. 4A represents a time-course response of MCT on doxombicin-treatedneutrophils.

FIG. 4B represents a time-course response of doxorubicin on MCT-treatedneutrophils.

FIG. 5 shows the effect of MCT and tricaprin on bone marrowproliferation.

FIG. 6 shows the effect of MCT on bone marrow cell count inimmunosuppressed animals.

FIG. 7 shows the effect of MCT on spleen cell count in immunosuppressedanimals.

FIG. 8 shows the effect of MCT and GM-CSF on thymus weight on normalmice.

FIG. 9 shows the effect of MCT, sodium caprylate and sodium caprate onbone marrow cell count in immunosuppressed animals.

FIG. 10 represents the chemoprotective effect and anti-tumor efficacy ofMCT in combination with a sub-therapeutic concentration of doxorubicinin B16F10 melanoma model.

FIG. 11 represents the chemoprotective effect and anti-tumor efficacy ofMCT in combination with a sub-therapeutic concentration ofcyclophosphamide or taxotere in DA-3 breast carcinoma model.

FIG. 12 shows the chemoprotective effect and anti-tumor efficacy of MCTin combination with a therapeutic concentration of cyclophosphamide ortaxotere in DA-3 breast carcinoma model.

DETAILED DESCRIPTION OF THE INVENTION

High-dose chemotherapy and radiation destroy hematopoietic cells in bonemarrow, leaving the patients severely depleted in neutrophils andplatelets. After such treatments, patients spend several weeks inintensive care units due to infections and fever resulting fromneutropenia. Thrombocytopenia leads to prolonged clotting time andbleeding disorders requiring platelet transfusions. Myelosuppression isa dose-limiting factor in cancer treatment and lack of neutrophils andplatelets is the leading cause of morbidity and mortality followingthese cancer treatments.

In bone marrow transplantation, two approaches may be used. Beforetransplantation, stimulation of the bone marrow may increase the numberof peripheral progenitor stem cells. However, freshly transplanted bonemarrow does not contain sufficient mature neutrophils or neutrophilintermediaries to restore a patient's immune system. This leaves thepatient with a period of increased susceptibility to infections andprolonged clotting time. Therapy involving neutrophil stimulation andactivation increases recovery following bone marrow transplantation, byreducing neutropenia and thrombocytopenia.

The present invention relates to a method of promoting neutrophilsurvival and activation in a subject. Current methods are directedtowards restoring the subject's hematopoietic system. Hematopoieticgrowth factors presently used for such treatment are granulocytecolony-stimulating factor (G-CSF), stem cell factor (SCF) andgranulocyte-macrophage colony-stimulting factor (GM-CSF). G-CSF andGM-CSF can shorten the total period of neutropenia and thrombocytopeniabut there still remains a significant window during which the patient isdeficient in blood clotting and susceptible to infections.

In bone marrow transplantation, “mobilization” has also been used toharvest higher numbers of stem/progenitor cells from peripheral blood.Hematopoietic stem cells in the bone marrow are mobilized in the bloodfollowing treatment with growth factors. Growth factors used for suchtreatment include interleukin-3 (IL-3), G-CSF, GM-CSF, SCF and arecombinant fusion protein having the active moieties of both IL-3 andGM-CSF. Mobilized stem cells are then harvested after growth factortreatment and reinfused into the patient following the next round ofhigh dose chemotherapy or irradiation, to restore the patient'sneutrophils and platelets.

Medium-chain triglycerides (also referred to herein as “MCT”) consist ofglycerol esterified with fatty acids with carbon chain lengths of 8 (C8,octanoic acid or caprylic acid) and 10 (C10, decanoic acid or capricacid). MCT usually contain of a mixture of glycerol esters of C8 and C10fatty acids. However, MCT can also contain small amounts (2±1% each) ofglycerol esters of C6 (hexanoic acid or caproic acid) and C12(dodecanoic acid or lauric acid). CRODAMOL™ is a commercially availableMCT available from Croda Ltd., Toronto (Canada). As shown in example 1,CRODAMOL™ is an MCT which contains glycerol triesters of C8 and C10fatty acids present in varying proportions. However, CRODAMOL™ does notcontain any C6 or C12 fatty acid esters. Long-chain triglycerides (alsoreferred to herein as “LCT”), on the other hand, consist of glycerolesterified with fatty acids with carbon chain lengths of greater than12. Typical fatty acids present in LCT include palmitic (C16) andstearic (C18) acids. Unlike MCT, LCT is the primary component of dietaryfats. Indeed, MCT and LCT have significantly different biologicalproperties. Some of the physiological differences between MCT and LCTare described in Harrison's Principles of Internal Medicine, 8^(th)Edition, 1520-1521 (1977), McGraw Hill Book Company or 15^(th) Edition,1668-1669 (2001). For example, MCT, in contrast to LCT, do not requirehydrolysis by pancreatic lipase, since they can be absorbed by theintestinal epithelial cell.

MCT and their constituent medium-chain fatty acids are nontoxicmaterials which find use in the food and pharmaceutical industries. Forexample, K. A. Trani et al. in Food and Chemical Toxicology 38: 79-98(2000) state that MCT have been utilized in an increasing number of foodand nutrition applications because they offer a number of advantagesover LCT. MCT are also used primarily as emulsifiers in various humanand veterinary pharmaceutical preparations and in cosmetics. They referto a number of toxicological studies which support the safety of MCT.For example, they note that the safety of human dietary consumption ofMCT, up to levels of 1 g/kg, has been confirmed in clinical trials. C8and C10 fatty acids possess similar safety and use. For example, in TheMerck Index, 11^(th) Edition, 266 (1989) caprylic acid is reported tohave an LD₅₀ (oral, rats)=10.08 g/kg which is essentially nontoxic. Infact, according to section 184 of the Code of Federal Regulations (CFR),the U.S. Federal Drug Agency (FDA) has granted caprylic acid a GRAS(Generally Recognized As Safe) affirmation. Similarly, according tosection 172 (CFR) free fatty acids (e.g. capric, caprylic) and theirmetallic salts are recognized as safe additives for use in food. Asnoted by D. Dimitrijevic et al. in Journal of Pharmacy and Pharmacology53: 149-154 (2001), capric acid (sodium salt) is approved for human usein Japan and Sweden as an absorption enhancer for rectal drug products.U.S. Pat. No. 4,602,040 (1986) describes the use of MCT as apharmaceutical excipient. More recently, PCT publication WO 01/97799describes the use of medium-chain fatty acids, in particular caprylicand capric acids, as antimicrobial agents.

However, until the unexpected findings disclosed herein, theeffectiveness of medium-chain fatty acids such as capric acid, caprylicacid or metallic salts or mono-, di- or triglycerides (MCT) thereof as aneutrophil survival and activation factor was not known. As describedherein, MCT contain triglycerides of C8 (caprylic) and C10 (capric)fatty acids which constitute at least 98% of the activity pertaining tostimulation of hematopoiesis and maturation of neutrophils. D. Waitzberget al. in Nutrition 13: 128-132 (1997) state that lipid emulsions (LCTand MCT) only moderately decreases neutrophil bactericidal function andhave no effect on monocytes. Indeed, the only publication which gives avague indication that MCT may influence neutropenia describes clinicalstudies in which MCT are administered along with LCT and compared withLCT alone. No studies were undertaken with MCT alone and so the effecton immune function is not apparent. However, the results reported by S.Demirer et al. in Clinical Nutrition 19: 253-258 (2000) teaches that MCTexacerbate neutropenia when MCT are combined with LCT and relative toLCT alone. That is, it was suggested that MCT inhibit neutrophilfunction and/or survival. Somewhat supporting this suggestion, PCTpublication WO 95/30413 asserts that unsaturated long chain fatty acidssuch as linolineic acid, as well as saturated long chain (C16 or longer)fatty acids, can function to enhance hematopoietic stem cellproliferation.

The present invention relates to the use of medium-chain fatty acids ormetallic salts or triglycerides thereof or mono- or diglycerides orother analogues thereof or MCT as a hematopoiesis activation or growthfactor and a neutrophil survival and activation factor. When used inchemotherapy and radiotherapy, a composition containing medium-chainfatty acids or metallic salts or triglycerides thereof or mono- ordiglycerides or other analogues thereof or MCT is administered before,during and/or after the treatment in order to shorten the neutropenicwindow and to accelerate the replenishment of the hematopoietic system.Furthermore, it is possible to use a combination of medium-chain fattyacids along with their metallic salts or triglycerides thereof or mono-or diglycerides or other analogues thereof and/or MCT at multiple pointsrelative to treatment with chemotherapy and radiotherapy (e.g., fattyacids before treatment and MCT after). Alternatively, it is possible toadminister the combination simultaneously: before, during and/or aftertreatment with chemotherapy and radiotherapy. In severe neutropenia, acomposition containing medium-chain fatty acids or metallic salts ortriglycerides thereof or mono- or diglycerides or other analoguesthereof or MCT is used as the therapeutic agent. In bone marrowtransplantation, medium-chain fatty acids or metallic salts ortriglycerides thereof or mono- or diglycerides or other analoguesthereof or MCT is used to increase the number of peripheral stem cellsavailable for transplantation after ablative radiotherapy orchemotherapy. Medium-chain fatty acids or metallic salts ortriglycerides thereof or mono- or diglycerides or other analoguesthereof or MCT can also be used after bone marrow transplantation inorder to stimulate bone marrow stem cells thus shortening the timeperiod for recovery from neutropenia.

The method is therefore useful for stimulating hematopoiesis to treatmyelosuppression arising from chemotherapy or radiotherapy; chronic ortransient neutropenia neutropenia; drug-induced neutropenia; andneutropenia arising from a hematologic disease, nutritional deficiency,infection, or radiotherapy. Transient neutropenia may arise from stressdue to shipping of an animal or travel of a human or animal. The methodis also useful for stimulating hematopoiesis to heal a wound in thepatient, and to induce neutrophil mobilization to facilitate bone marrowtransplantation in a patient.

As used herein, the terms “a” or “an” can mean one or more, depending onthe context in which it is used.

As used herein, “medium-chain fatty acids such as capric acid orcaprylic acid or metallic salts or triglycerides thereof or mono- ordiglycerides or other analogues thereof or MCT composition” refers to acomposition comprising said active ingredient and one or morepharmaceutically acceptable carriers.

As used herein, the term “pharmaceutically acceptable carrier” refers toa substance that does not interfere with the physiological effects ofmedium-chain fatty acids such as capric acid or caprylic acid ormetallic salts or triglycerides thereof or mono- or diglycerides orother analogues thereof or MCT and that is not toxic to mammalsincluding humans.

The capric or caprylic acid or salts or triglycerides thereof or mono-or diglycerides or other analogues thereof or MCT compositions of thepresent invention are formulated using capric or caprylic acid or saltsor triglycerides thereof or mono- or diglycerides or other analoguesthereof or MCT and pharmaceutically acceptable carriers by methods knownto those skilled in the art (MERCK INDEX, Merck & Co., Rahway, N.J.).These compositions include, but are not limited to, liquids, oils,emulsions, aerosols, inhalants, capsules, pills, patches andsuppositories.

All methods include the step of bringing the active ingredient(s) intoassociation with the carrier which constitutes one or more accessoryingredients.

As used herein, the term “chemotherapy” refers to a process of killingproliferating cells using a cytotoxic agent. The phrase “during thechemotherapy” refers to the period in which the effect of theadministered cytotoxic agent lasts. On the other hand, the phrase “afterthe chemotherapy” is meant to cover all situations in which acomposition is administered after the administration of a cytotoxicagent regardless of any prior administration of the same and alsoregardless of the persistence of the effect of the administeredcytotoxic agent.

When the method of this invention is applied to chemotherapy, a capricor caprylic acid or salts or triglycerides thereof or mono- ordiglycerides or other analogues thereof or MCT composition can beadministered prior to, during, or subsequent to the chemotherapy (i.e.,prior to, during, or subsequent to the administration of a cytotoxicagent).

By “cytotoxic agent” is meant an agent which kills highly proliferatingcells, e.g., tumor cells, virally infected cells, or hemopoietic cells.Examples of a cytotoxic agent which can be used to practice theinvention include, but are not limited to, cyclophosphamide,doxorubicin, daunorubicin, vinblastine, vincristine, bleomycin,etoposide, topotecan, irinotecan, taxotere, taxol, 5-fluorouracil,methotrexate, gemcitabine, cisplatin, carboplatin or chlorambucil, andan agonist of any of the above compounds. A cytotoxic agent can also bean antiviral agent, e.g., AZT (i.e., 3′-azido-3′-deoxythymidine) or3TC/lamivudine (i.e., 3-thiacytidine).

As used herein, the term “leukopenia” refers to an abnormal reduction inthe number of leukocytes in the blood.

As used herein, the term “neutropenia” refers to the presence ofabnormally small numbers of neutrophils in the blood.

In one preferred embodiment, the pharmaceutical composition is in theform of any suitable composition for oral, sublingual administration orinhalation (nasal spray), intravenous, intramuscular, subcutaneous, foruse in the treatment of neutropenia, thrombocytopenia or as a neutrophilsurvival and activation factor.

It will be appreciated that the amount of a composition of the inventionrequired for use in the treatment will vary with the route ofadministration, the nature of the condition being treated, the age andcondition of the patient, and will be ultimately at the discretion ofthe attendant physician. The desired dose may conveniently be presentedin a single dose or as divided doses taken at appropriate intervals, forexample as two, three, four or more doses per day.

While it is possible that, for use in therapy, medium-chain fatty acidsor metallic salts or triglycerides thereof or mono- or diglycerides orother analogues thereof or MCT may be administered as the raw chemical,it is preferable to present the active ingredient as a pharmaceuticalformulation.

In a preferred embodiment of this invention, the amount of activeingredient administered is such that the concentration in the blood(free and/or bound to serum albumin) is greater than 1 μM. In aparticularly preferred embodiment, the concentration in the blood isgreater than 1 mM.

In another embodiment, the pharmaceutical composition is in the form oforal (including sublingual), or parental (including intramuscular,subcutaneous rectal and intravenous) administration. The formulationsmay, where appropriate, be conveniently presented in discrete dosageunits and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing into association theactive compound with liquid carriers or finely divided solid carriers orboth and then, if necessary, shaping the product into the desiredformulation. When desired, the above-described formulations adapted togive sustained release of the active ingredient may be employed.

Medium-chain fatty acids or salts or triglycerides thereof ormono-diglycerides or other analogues thereof or MCT can also be used incombination with other therapeutically active agents such as cytotoxicanticancer agents or other anticancer agents (immune modulating orregulating drugs or therapeutic vaccines or antiangiogenesis drugs,etc.), immune suppressive drugs (including anti-inflammatory drugs), agrowth factor such as a colony stimulating factor (preferably GM-CSF orG-CSF), a cytokine such as interleukin 2 or interleukin 15, orcombinations thereof. The individual components of such combinations maybe administered either sequentially (before or after) or simultaneouslyin separate or combined pharmaceutical formulations. The combinationreferred to above may conveniently be presented for use in the form of apharmaceutical formulation and thus pharmaceutical formulationscomprising a combination as defined above together with apharmaceutically acceptable carrier thereof comprise a further aspect ofthe invention.

In a preferred embodiment of the method of stimulting hematopoiesis in apatient needing treatment, a pharmacologically effective amount of acomposition containing one or more of the following compounds, orcombinations thereof, are administered:

whereinR₁ is a straight chained or branched, saturated or unsaturated C7-C11alkyl group;A and B are hydrogen or

independently; andX is a hydroxyl group, an oxy anion with a metallic mono- or dicationiccounterion, or an alkoxy group with a straight chained or branched C1-C4alkyl moiety.

It will be understood by those skilled in the art that, in formula III,the term “Z=zero” indicates that the variable Z is optional and may beeliminated or replaced with a hydrogen.

In an alternative preferred embodiment, the composition contains amixture of at least two compounds described by formula I, which areMedium Chain Triglycerides (MCTs) wherein A, B and R₁ are the same andare straight chained or branched, saturated or unsaturated C7 and C9alkyl groups, respectively. Alternatively, the composition contains amixture of two triglycerides wherein a first MCT is described by formulaI, wherein A, B and R₁ are CH₃(CH₂)₆, and a second MCT is described byformula I, wherein A, B and R₁ are CH₃(CH₂)₈. Alternatively, thecomposition further contains from 0.1% to 3% each of a third compounddescribed by formula I, wherein A, B and R₁ are CH₃(CH₂)₄, and a fourthcompound described by formula I, wherein A, B and R₁ are CH₃(CH₂)₁₀.Alternatively, the composition is a mixture containing four geometricisomers of C8 and C10 fatty acid triglycerides described by thefollowing formula:

In an alternative preferred embodiment, the composition contains one ormore compounds described by formula II or formula III, wherein X is OHor X is an oxy anion with a metallic counterion such as calcium,magnesium, potassium, and sodium.

In a more preferred embodiment, the composition is caprylic acid, capricacid, sodium caprylate, sodium caprate, calcium caprylate, calciumcaprate, caprylic acid triglyceride, or capric acid triglyceride.

The compositions and methods described herein include the followinganalogues and compounds:

-   aza analogues of caprylic acid triglyceride or capric acid    triglyceride, preferably where the aza analogue is    1,2,3-O,N,O-trioctanoyl serinol or 1,2,3-O,N,O-tridecanoyl serinol;-   the compound described by formula IV

-   the compound described by formula V

-   the compound described by formula VI, which provide a pharmaceutical    formulation by degradation in vivo to release active substances    described above

The following examples further illustrate the practice of thisinvention, but are not intended to be limiting thereof. It will beappreciated that the selection of the dose of medium-chain fatty acidsor salts or triglycerides thereof or mono- or diglycerides or otheranalogues thereof or MCT and related pharmaceutical formulations to beadministered to any individual patient (human or animal) will fallwithin the discretion of the attending physician, will be prescribed ina manner commensurate with the appropriate dosages and will depend onthe stage of the disease and like factors uniquely within the purview ofthe attending physician.

Example 1 Analysis of CRODAMOL™ (MCT: Caprylic/Capric Triglyceride)

CRODAMOL™ GTCC lot #T1033-1299 from Croda Ltd. (Toronto, Canada) wasanalyzed by gas chromatography. GC FID-analysis, conditions of thegradient: 100° C.-250° C. in 10 minutes, then 250° C. for 25 minutes;FID 250° C. Four peaks were observed: 22.04 minutes (26%), 25.07 minutes(43%), 29.16 minutes (25%) and 34.75 minutes (5%).

A sample of caprylic triglyceride (tricaprylin), obtained fromSigma-Aldrich lot #079H1212, was analyzed by gas chromatography. GCFID-analysis, conditions of the gradient: 100° C.-250° C. in 10 minutes,then 250° C. for 25 minutes; FID 250° C. Mainly one peak at 22.31minutes (98%).

Example 2 Acylation of Alcohol Using Acid Chloride and Pyridine Base

General Method A (Pyridine)

A solution of the alcohol (˜0.1 M) in dry CH₂Cl₂ and pyridine (4:1), wascooled to 0° C. under nitrogen, and treated with the acid chloride (1.2equivalent). The reaction was allowed to warm slowly to ambienttemperature, and stirred overnight. TLC analysis (SiO₂, EtOAc 1:9hexane) showed no remaining alcohol. The reaction mixture was dilutedwith CH₂Cl₂, and washed with saturated aqueous NH₄Cl solution. Theaqueous phase was extracted with CH₂Cl₂ (×1) and hexane (×1), andcombined organic phases were washed with saturated aqueous NaClsolution, dried over Na₂SO₄, filtered and evaporated in vacuo to givethe crude product.

General Method B (DMAP)

A solution of the alcohol (˜0.1 M) in dry CH₂Cl₂ was cooled to 0° C.under nitrogen, and treated with DMAP (1.3 equivalent) and the acidchloride (1.2 equivalent). The reaction was allowed to warm slowly toambient temperature, and was stirred overnight. TLC analysis (SiO₂,EtOAc 1:9 hexane) showed no remaining alcohol. The reaction mixture wasdiluted with CH₂Cl₂, and washed with saturated aqueous NH₄Cl solution.The aqueous phase was extracted with CH₂Cl₂ (xl) and hexane (xl), andcombined organic phases were washed with saturated aqueous NaClsolution, dried over Na₂SO₄, filtered and evaporated in vacuo to givethe crude product.

Example 3 Nonanoic Acid Triglyceride

Glycerol (120 mg, 1.30 mmol) was acylated with nonanoyl chloride (751μl, 4.16 mmol) according to General Method A, example 2. Purification bycolumn chromatography (Isolute™ SiO₂, eluting with 0-5% EtOAc in hexane)gave two product containing fractions, which were evaporated in vacuo togive the desired product as a colourless liquid, in 89% (127 mg, 19%)and 93% (475 mg, 71%) purity respectively (GC/FID). R_(f) 0.46 (SiO₂,10% ethyl acetate in hexane); ¹H NMR (CDCl₃, 300 MHz) δ_(H)=5.27 (m,1H), 4.29 (dd, 2H), 4.14 (dd, 2H), 2.31 (m, 6H), 1.61 (m, 6H), 1.27 (m,30H), 0.88 (t, 9H); MS (FAB⁺) m/z=510(M−H⁺); GC FID-analysis,conditions: gradient 100° C.-250° C. in 10 minutes, then 250° C. for 25minutes; FID 250° C.; 27.25 minutes.

Example 4 Nonanoic acid Diglyceride and Monoglyceride

Glycerol (100 mg, 1.09 mmol) was acylated with one equivalent ofnonanoyl chloride (205 μl, 1.09 mmol) according to General Method A,example 2. Purification by Biotage™ (40S, SiO₂, eluting with 10% ethylacetate in hexane-100% ethyl acetate) gave a colorless oil. Twodifferent compounds were obtained:

Nonanoic acid Diglyceride was obtained (73 mg, 18%) as a white solid. mp24-26° C.; R_(f) 0.52 (SiO₂ pretreated with Et₃N, 30% ethyl acetate inhexane); ¹H NMR (CDCl₃, 300 MHz) δ_(H)=4.17 (m, 5H), 2.35 (t, 4H), 1.63(m, 4H), 1.27 (m, 20H), 0.88 (t, 6H); MS (FAB⁺) m/z=373 (M+H⁺).

Nonanoic acid Monoglyceride was obtained (85 mg, 34%) as a white solid.mp 37-38.5° C.; R_(f) 0.08 (SiO₂ pretreated with Et₃N, 30% ethyl acetatein hexane); ¹H NMR (CDCl₃, 300 MHz) δ_(H)=4.18 (m, 2H), 3.94 (m, 1H),3.69 (m, 1H), 3.62 (m, 1H), 2.36 (t, 2H), 1.62 (m, 2H), 1.28 (m, 10H),0.88 (t, 3H); MS (FAB⁺) m/z=233 (M+H⁺).

Example 5 1,2,3-O,N,O-Tridecanoyl serinol

Serinol (51 mg, 0.56 mmol) was acylated with decanoyl chloride (372 μl,1.76 mmol) according to General Method B, example 2. Purification byMPLC (SiO₂, eluting with 0 then 10% EtOAc in hexane) gave the desiredproduct as a white solid (301 mg, 97%). mp 54° C.; TLC, R_(f) 0.85(SiO₂, EtOAc 2:3 hexane); ¹H NMR (CDCl₃, 300 MHz) δ_(H) 0.84 (9H, t),1.20-1.27 (36H, m), 1.52-1.60 (6H, m), 2.13 (2H, t), 2.28 (4H, t), 4.03(2H, 2×A of 2×ABX), 4.19 (2H, 2×B of 2×ABX,), 4.41-4.46 (1H, m), 5.70(1H, d); HRMS m/e calcd for C₃₃H₆₃NO₅ 553.4706 Found 553.4713. GC-FIDanalysis, conditions of the gradient: 100° C.-250° C. in 10 minutes,then 250° C. for 25 minutes; FID 250° C. Mainly one peak at 14.80minutes (98%).

Example 6 1,3-O,O-Didecanoyl serinol

A solution of serinol (1.57 gm, 17.2 mmol) in acetone (17 ml) and water(17 ml), was treated with triethylamine (3.60 ml, 25.9 mmol) and BOC—ON(4.67 gm, 19.0 mmol), and the reaction was stirred under nitrogenovernight. Acetone was evaporated in vacuo, and the crude mixture waspartitioned between EtOAc and water. The aqueous phase was extractedwith EtOAc (×3), and combined organic extracts were dried over Na₂SO₄,filtered and evaporated in vacuo, to give a yellow solid. Purificationby HPLC (SiO₂, eluting with 40 to 80% EtOAc in hexane) gave theN—BOC-diol intermediate as a white crystalline solid (2.10 gm, 64%).TLC, R_(f) 0.15 (SiO₂, EtOAc 4:1 hexane); ¹H NMR (CDCl₃, 300 MHz) δ_(H)1.40 (9H, s), 3.54-3.56 (5H, m).

The N—BOC-diol intermediate (50 mg, 0.26 mmol) was acylated withdecanoyl chloride (173 μl, 0.83 mmol) according to General Method B.Purification by MPLC (SiO₂, eluting with 0 then 10% EtOAc in hexane)gave the N—BOC-diacyl intermediate as a colourless oil (115 mg, 88%).TLC, R_(f) 0.80 (SiO₂, EtOAc 2:3 hexane); ¹H NMR (CDCl₃, 300 MHz) δ_(H)0.87 (6H, t), 1.23-1.30 (24H, m), 1.44 (9H, s), 1.56-1.70 (4H, m), 2.31(4H, t), 4.04-4.21 (4H, m), 4.77-4.80 (1H, m), 6.73 (1H, d).

A solution of the N—BOC-diacyl intermediate (76 mg, 0.15 mmol) in dryCH₂Cl₂ (1.5 ml) was cooled to 0° C., and treated with a solution of 4.0M anhydrous HCl in 1,4-dioxane (375 μl, 1.50 mmol; final concentration0.8 M). The reaction was allowed to warm to room temperature, andstirred for 3 hr at the same temperature. A further portion of 4.0 Manhydrous HCl in 1,4-dioxane (375 μl, 1.50 mmol) was added, and thereaction was stirred for a further 2 hr. Evaporation of solvents gavethe desired product, as a white solid (69 mg, 100%). mp 101° C.; TLC,R_(f) 0.40 (SiO₂, EtOAc 2:3 hexane); ¹H NMR (CDCl₃, 300 MHz) δH 0.88(6H, t), 1.20-1.29 (24H, m), 1.55-1.65 (4H, m), 2.45-2.52 (4H, m),3.72-3.80 (1H, m), 4.30-4.51 (2H, m), 8.6-9.0 (3H, br m); HRMS m/e calcdfor (M−HCl), C₂₃H₄₅NO₄ 339.3348 Found 339.3340; GC-FID analysis,conditions of the to gradient: 100° C.-250° C. in 10 minutes, then 250°C. for 25 minutes; FID 250° C. Mainly one peak at 17.14 minutes (94%).

Example 7 α- and β-O-Methyl-2,3,4,—O,O,O-tridecanoyl-L-fucopyranose

1-O-Methyl-L-fucopyranose (593 mg, 3.33 mmol) was synthesised accordingto the method of Levene & Muskat (J. Biol. Chem. 105:431-441, 1934) andwas acylated with decanoyl chloride (2.90 ml, 14.0 mmol) according toGeneral Method B, example 2. Purification by MPLC (SiO₂, eluting with 0to 5% EtOAc in hexane) gave the α (1.18 gm, 55%) and β (0.52 gm, 24%) ofthe desired product as a colourless oil.

Data for α anomer: TLC, R_(f) 0.45 (SiO₂, EtOAc 1:9 hexane); ¹H NMR(CDCl₃, 300 MHz) δ_(H) 0.87 (9H, t), 1.14 (3H, d), 1.20-1.35 (36H, m),1.52-1.68 (4H, m), 2.18 (2H, t), 2.29 (1H, A of ABX₂), 2.32 (1H, B ofABX₂), 2.41 (2H, t), 3.38 (3H, s), 4.13 (1H, qd, J 6.5), 4.93 (1H, d),5.15 (1H, dd, 5.30 (1H, dd), 5.36 (1H, dd); FIRMS m/e calcd for(M-CH₃O)C₃₆H₆₅O₇ 609.4730 Found 609.4720.

Data for β anomer: TLC, R_(f) 0.40 (SiO₂, EtOAc 1:9 hexane); ¹H NMR(CDCl₃, 300 MHz) δ_(H) 0.87 (9H, t, J=6.5 Hz), 1.22 (3H, d), 1.20-1.35(36H, m), 1.49-1.67 (4H, m), 2.18 (2H, t), 2.25 (1H, A of ABX₂), 2.29(1H, B of ABX₂), 2.34 (2H, t), 3.50 (3H, s), 3.81 (1H, qd), 4.35 (1H,d), 5.03 (1H, dd), 5.19 (1H, dd), 5.24 (1H, dd).

Example 8 L-Glutamate Capramide

To a solution of capric acid (7.30 mmol, 1.26 g) in dry CH₂Cl₂ (60 ml)was added under nitrogen L-glutamic acid di-t-butyl ester HCl salt (6.09mmol, 1.80 gm), DMAP (1.8 mmol, 0.22 g), diisopropylethylamine (18 mmol,3 ml) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl salt (EDCI)(7.30 mmol, 1.40 gm). The resulting colorless solution was stirred atroom temperature for 24 hr. The solvent was then removed under reducedpressure to give a white oily residue. Purification by Biotage™ (40SSiO₂, eluting with 5% ethyl acetate in hexane-30% ethyl acetate inhexane) gave a colorless oil, which was L-glutamate di-t-butylestercapramide (2.47 gm, 98%). R_(f) 0.56 (SiO₂, 30% ethylacetate in hexane);¹H NMR (CDCl₃, 300 MHz) δ_(H)=6.05 (d, 1H), 4.45 (m, 1H), 2.30 (m, 2H),2.27 (m, 2H), 2.16 (t, 2H), 2.07 (m, 1H), 1.87 (m, 1H), 1.58 (m, 2H),1.43 (s, 9H), 1.41 (s, 9H), 1.23 (m, 12H), 0.84 (t, 3H).

Deprotection of the BOC group was achieved by a slow addition of asolution 4.0 M HCl in 1,4-dioxane (23 ml) to a solution of thedi-t-butyl ester derivative (5.75 mmol, 2.38 gm) in CH₂Cl₂ (35 ml) at 0°C. The colorless solution was allowed to warm to room temperature andstirred for an additional 20 hr. The solvent was then removed underreduced pressure and the resulting white solid was dried to yieldL-glutamate capramide (1.71 gm, 99%). mp 95-96.5° C.; ¹H NMR (CD₃OD, 300MHz) δ_(H)=4.39 (m, 1H), 3.27 (d, 1H), 2.36 (t, 2H), 2.20 (t, 2H), 2.13(m, 1H), 1.90 (m, 1H), 1.58 (m, 2H), 1.27 (m 12H), 0.86 (t, 3H); MS(ES⁺) m/z=324 (M+Na⁺), 302 (M+H⁺); MS (ES) m/z=300 (M−H⁺); HPLCanalysis, conditions: gradient 0.01% TFA in 10%-70% acetonitrile in 10minutes; flow 1.0 ml/min; 210 nm; 8.93 minutes.

Example 9 Capric Acid N,N-dimethylacetamide ester

To a solution of capric acid (8.7 mmol, 1.5 g) in anhydrous DMF (80 ml)under nitrogen was added sodium iodide (0.87 mmol, 130 mg) followed bydimethylchloroacetamide (9.6 mmol, 985 μl). Potassium carbonate (9.6mmol, 1.3 g) was then added and the resulting suspension stirred at 90°C. for 5 days. The reaction was allowed to cool at room temperature, andthen was mixed with distilled water. The product was extracted withethyl acetate (x3). The combined organic phases were washed with aqueoussolution of NaHCO₃, dried with Na₂SO₄, filtered, and concentrated underreduced pressure. The yellow liquid obtained was purified by Biotage™(40M, SiO₂, eluting with 25% ethyl acetate in hexane-50% ethyl acetatein hexane). Capric acid N,N-dimethylacetamide ester was obtained (2.03gm, 92%) as a white powder. mp 42-42.5° C.; R_(f) 0.55 (SiO₂, ethylacetate); ¹H NMR (CDCl₃, 300 MHz) δ_(H)=4.64 (s, 2H), 2.92 (s, 3H), 2.91(s, 3H), 2.38 (t, 2H), 1.62 (qt, 2H), 1.22 (m, 12H), 0.83 (t, 3H); MS(ES⁺) m/z=537 (2M+Na⁺), 280 (M+Na⁺), 258 (M+H⁺).

Example 10 In Vitro Assays of Neutrophil Apoptosis and Survival

Neutrophil survival was measured as described by Lagraoui and Gagnon(Cell. Mol. Biol. 43:313-318, 1997). Neutrophils were obtained from theperipheral blood of healthy volunteers. Blood was submitted to gradientcentrifugation with Lympholyte-poly (Cedarlane, Hornby, Canada) followedby hypotonic lysis of contaminating erythrocytes. Cells were suspendedin RPMI (Gibco, Burlington, Canada) supplemented with 10% FBS (Hyclone,Logan USA). Final cell preparations consisted of >95% neutrophils asdetermined by Wright Giemsa staining. Viability was greater than 97% asdetermined by trypan blue exclusion. Polymorphonuclear leukocytes (PMN)have a short half-life and rapidly undergo characteristic changesindicative of apoptosis. Apoptosis was assessed according to the methoddescribed by Nicoletti et al., J. Immunol. Meth. 139:271-279 (1991).Briefly, freshly isolated neutrophils were incubated for 24 hr at 37° C.with different concentrations of MCT. After incubation, cells werestained with propidium iodide (PI, Sigma) and analyzed for apoptosisusing an XL Flow Cytometer (Coulter). Data were then expressed as thepercent of apoptotic cells. FIG. 1 represents a compilation of severalexperiments in which neutrophil apoptosis was measured in the absence(control) or the presence of various concentrations of MCT. The resultsindicate that in the presence of MCT in vitro, neutrophil apoptosis isinhibited by up to 90% and that the inhibition is dose-dependent. Thus,MCT can increase neutrophil survival and can be used as a neutrophilsurvival factor.

Example 11 In Vitro Assays of PMN Phagocytosis

Neutrophils (2×10⁶/ml) were incubated for 24 hr, at 37° C. in 5% CO₂ and95% humidity with various concentrations of MCT. After 24 hr, viabilitywas determined by trypan blue exclusion and cells were washed threetimes with PBS containing 2 mM glucose, 1 mM MgCl₂ and 1 mM CaCl₂. Thecell concentration was then adjusted to 1×10⁶ cells/nil and thenincubated with fluoresbrite carboxylate microspheres (1/10 dilution).After 30 minutes of incubation, neutrophils were washed and fixed in 2%paraformaldehyde. Fixed neutrophils were analyzed for microspheresingestion using XL Flow Cytometer (Coulter). Data were then expressed asthe percent of phagocytic cells.

FIG. 2 represents a compilation of several experiments which measure PMNphagocytic activity in the absence (control) or the presence of variousconcentrations of MCT. The results indicate that MCT enhances thephagocytic activity of human PMN. The phagocytic activity is enhanced upto two to three times from the control values and the extent of thestimulation depends on the immune status of the donor.

Example 12 Effect of Doxorubicin on Neutrophil Apoptosis

PMN were isolated as described in example 10. Cells (2×10⁶/ml) wereincubated for 4 hr, at 37° C. in 5% CO₂ and 95% humidity in the presenceof various concentrations of a chemotherapeutic agent, doxorubicin.Apoptotic cells were evaluated as described in example 10. Data areexpressed in percent of apoptotic cells. FIGS. 3A and 3B indicate thatdoxorubicin induces PMN apoptosis.

Example 13 MCT Rescues the Doxorubicin-Induced Apoptosis of Neutrophils

PMN were isolated as described in example 10. Cells (2×10⁶/ml) wereincubated for 4 hr, at 37° C. in 5% CO₂ and 95% humidity in the presenceof various concentrations of doxorubicin with or without MCT (2.5% and5.0%). Apoptotic cells were evaluated as described in example 10. Dataare expressed in percent of apoptotic cells.

Table 1 represents two experiments measuring the chemoprotective effectsof MCT on PMN. Results are expressed in percent of apoptotic cells after4 hr of incubation in the presence or the absence of doxorubicin with orwithout MCT. As in example 12, doxorubicin induces PMN apoptosis invitro. However, in the presence of MCT, at a concentration of 2.5% and5% (v/v), the apoptotic effects of doxorubicin are inhibited. Thus, MCTexerts an anti-apoptotic action on PMN. Apoptosis was also studied usingthe annexin V-FITC/PI (propidium iodide) to method according to themanufacturer's Biosources recommendations (Apotarget Annexin-VFITCApoptosis Kit #PHN 1018). Annexin V binds to phosphatidylserine which istransferred from the internal to external membrane in early to latephase apoptosis. Briefly, neutrophils are incubated in the presence orabsence of varying concentrations of doxorubicin and MCT. After 24 hr,neutrophils are washed with PBS and stained with 2 μA of Annexin V-FITCand 10 μl of PI (Sigma, 1 mg/ml) for 20 minutes. After incubation,stained cells were fixed in paraformaldehyde (1%) and analyzed forapoptosis using an XL Flow Cytometer (Coulter). Data were then expressedas the percent of apoptotic cells. FIG. 4A represents a time-courseresponse of MCT on doxorubicin-treated neutrophils. MCT rescues humanneutrophil doxorubicin-induced apoptosis in a time- and dose-dependentmanner.

FIG. 4B represents a time-course response of doxorubicin on MCT-treatedneutrophils. MCT protects, in a dose-dependent manner, neutrophilsagainst doxorubicin-induced apoptosis up to 4 hr before the introductionof the toxic agent (doxorubicin).

TABLE 1 Protective effect of MCT on doxorubicin- induced neutrophilapoptosis % Apoptosis of neutrophils (PMN) Experiment 1 Experiment 2 MCTMCT MCT MCT Doxorubicin 2.5% 5% 2.5% 5% Concentration Control (v/v)(v/v) Control (v/v) (v/v) 0 12.4 9.5 12.3 49.6 23.6 4.6 10⁻¹⁰ M  9.311.7 7.4 60.6 14.7 23.9 10⁻⁸ M 18.7 14.2 8.3 59.2 32.5 14.8 10⁻⁶ M 23.212.7 3.5 55.0 21.6 16.9 10⁻⁵ M 23.8 12.3 8.3 66.2 74.7 12.1 10⁻⁴ M 27.535.2 17.1 53.2 58.6 55.7

Example 14 MCT Rescues the Doxorubicin-Induced Apoptosis of NeutrophilsComparison to GM-CSF

Table 2 represents the effect of GM-CSF, MCT and tricaprylin ondoxorubicin-induced human neutrophil apoptosis. GM-CSF and MCT are ableto rescue or protect human neutrophils against doxorubicin-inducedapoptosis. Tricaprylin rescues doxorubin-induced apoptosis and furtherenhances the viability of human neutrophils to a higher extent than thatobserved in the non-treated neutrophils (control, absence ofdoxorubicin).

TABLE 2 Protective effect of MCT and GM-CSF on doxorubicin-inducedneutrophil apoptosis % PMN Viability Control  35.9 ± 0.71 Doxorubicin(DOX) (10⁻⁵ M) 6.82 ± 0.5 GM-CSF (10⁻⁷ M) + DOX 16.75 ± 2.05 GM-CSF(10⁻⁸ M) + DOX  6.99 ± 0.23 MCT (24 mM) + DOX 14.20 ± 1.98 MCT (12 mM) +DOX 12.37 ± 1.72 Tricaprylin (24 mM) + DOX 12.13 ± 1.25 Tricaprylin (12mM) + DOX 42.95 ± 6.15

Example 15 MCT and Tricaprin Increase In Vitro Murine Bone MarrowProliferation

Bone marrow cells were obtained from the femur of female C57BL/6 mice(6- to 8-weeks old). Cells were flushed and washed with PBS. Collectedcells are centrifuged and resuspended at 2×10⁶ cells/ml. 100 μl of cells(2×10⁵ cells) are incubated in a 96-well microliter plate for 48 hr inthe presence or absence of MCT or tricaprin. After incubation, cells arepulsed with 1 μCi of [³H]-thymidine for 6 hr. Plates are harvested on aTomteck and counted on a Microbeta β-counter. Incorporation of[³H]-thymidine in the DNA is a direct indication of the to cellproliferation.

FIG. 5 represents a typical experiment on the effect of MCT andtricaprin on bone marrow proliferation. MCT and tricaprin increase bonemarrow proliferation by 3- to 5-fold relative to the control.

Example 16 Chemoprotection Studies: In Vivo Induction of Immune CellsProliferation or Protection by MCT

Female C57BL/6 mice, 6 to 8 week old, were immunosuppressed by treatmentwith 80 mg 5-fluorouracil (5-FU) or 100-200 mg of cyclophosphamide (CY)or 12 mg of taxotere (TX) administered intravenously at day 0. Toexamine the immunoprotective effect of MCT or other compounds, mice werepre-treated orally at day-3, -2 and -1 or treated intravenously at day 0with the test compound. Mice were sacrificed at day +5 by cardiacpuncture and cervical dislocation. Then, cell suspensions were preparedfrom thymus, spleen and bone marrow as follow.

Tissues were crushed in PBS buffer and contaminating erythrocytes werelysed in ACK buffer (155 mM NH₄Cl, 12 mM NaHCO₃, 0.1 mM EDTA, pH 7.3)for 5 minutes. Cells were then collected by centrifugation and washedthree times in PBS and resuspended in tissue culture medium. Cells werecounted on a hemacytometer.

Results show that MCT significantly increases the number of cells inimmune tissues of normal and immunosuppressed animals compared to thevehicle alone as shown in the following tables and figures. Depending onthe experiments and the immune status of the mice, MCT can increase thebone marrow cell and/or the spleen cell and/or the thymus cell counts.

FIG. 6 shows the effect of MCT on bone marrow cell count inimmunosuppressed animals. Only CY and 5-FU depressed the bone marrowcell count compared to the control (no cytotoxic treatment). In mouse,taxotere treatment has no significant effect on bone marrow cell count.In suppressed bone marrow, administration of MCT (6.25 μMole per mouse)at day-3, -2 and -1 enhanced significantly the bone cell marrow count.

FIG. 7 represents the effect of MCT on spleen cell count inimmunosuppressed mice which received the pre-treatment regimen of MCTper o.s. All cytotoxic drugs (CY, 5-FU and TX) reduce significantly thespleen cell count compared to the control. Administration of MCT (6.25μMole per mouse) at day -3, -2 and -1 significantly increases spleencell count with “P” less than 0.0017, 0.009 and 0.0036 for CY, 5-FU andTX respectively.

Furthermore, MCT significantly enhances bone marrow cell count in normalmice when administered i.v. at day 0 (table 3). However, one i.v.injection of MCT is not sufficient to improve the spleen cell count inboth normal and immunosuppressed mice.

TABLE 3 Effect of cyclophosphamide (CY) and CY + MCT on bone marrow andspleen cells (normal mice) Bone Marrow Spleen # Cells (×10⁶) P # Cells(×10⁶) P Control 16 ± 3.94 94 ± 11 CY 13 ± 3.92 0.17 60 ± 12 0.0014 CY +MCT 17 ± 4.28 0.87 53 ± 10 0.0003 (50 μMole) CY + MCT 17 ± 6.15 0.95 51± 10 0.0002 (12.5 μMole) MCT (50 μMole) 41 ± 6.11 >0.0001 103 ± 7  0.19MCT (12.5 μMole) 27 ± 4.19 0.0018 101 ± 11  0.31

Example 17 Chemoprotection Studies: In Vivo Dose-Response of MCTInduction of Immune Cell Proliferation when Administered at Day-3, -2and -1 In Normal Mice

In vivo dose-response of MCT induction of immune cell proliferation innormal mice was assessed by the protocol described in example 16.

Table 4 represents the dose-response on treatment of MCT orallyadministered at day -3, -2 and -1 in normal mice. MCT significantlyincreases the bone marrow and spleen cell counts.

TABLE 4 Effect of MCT on normal mice Bone Marrow Spleen # Cells (×10⁶) P# Cells (×10⁶) P Control 45 ± 7.3 120 ± 12.9 MCT (3.15 μMole) 52 ± 4.30.10 144 ± 15.8 0.018 MCT (6.25 μMole)  59 ± 11.3 0.05 134 ± 13.9 0.129MCT (12.5 μMole) 54 ± 6   0.04 144 ± 19.8 0.04 MCT (25 μMole) 56 ± 3.90.01 127 ± 17.0 0.48

Example 18 Chemoprotection studies: In Vivo Induction of Immune CellsProliferation or Protection: Comparison of the Effect of MCT VersusGm-CSF

In vivo comparison on the induction of immune cellproliferation/regeneration or protection was undertaken following theprotocol described in example 16. Comparative studies of MCT and GM-CSFwere performed on normal and immunosuppressed animals. Compared to MCT,GM-CSF has no significant activity on bone marrow and spleen cell countsin immunosuppressed animals. A significant effect of GM-CSF was observedonly on thymus weight in normal mice (FIG. 8). In this case, MCTdisplayed a similar effect to GM-CSF.

Example 19 Chemoprotection Studies

The effect of caprylic acid and capric acid on in vivo induction ofimmune cell proliferation or protection was assessed by the protocoldescribed in example 16. As shown in table 5, only capric acidsignificantly enhances the bone marrow cell counts. No significanteffect was demonstrated on spleen cell counts, compared tocyclophosphamide treated mice.

TABLE 5 Effect of cyclophosphamide (CY), CY + caprylic acid and CY +capric acid on bone marrow and spleen cells Bone Marrow Spleen # CellsP/ # Cells P/ (×10⁶) Control P/CY (×10⁶) Control P/CY Control 54 ± 5.9 66 ± 7.3 CY 22 ± 5.7  >0.0001 23 ± 4.0 >0.0001 CY + 26 ± 3.58 0.001 0.2128 ± 6.4 >0.0001 0.17 caprylic acid CY + 32 ± 2.71 0.0004 0.006 27 ±8.4 >0.0001 0.27 capric acid

Example 20 Chemoprotection Studies

The effect of tricaprylin and tricaprin on in vivo induction of immunecell proliferation or protection assessed by the protocol described inexample 16. Tricaprylin and tricaprin are both efficacious in theproliferation or protection of bone marrow cell counts in CY-treatedmice (table 6). No significant effect was observed on the spleen cellcount, compared to cyclophosphamide treated mice.

TABLE 6 Effect of cyclophosphamide (CY), CY + tricaprylin and CY +tricaprin on bone marrow and spleen cells Bone Marrow Spleen # Cells P/# Cells P/ (×10⁶) Control P/CY (×10⁶) Control P/CY Control 55 ± 9.3 113± 15.9  CY 22 ± 5.8 0.0001 36 ± 13.6 >0.0001 CY + 34 ± 7.8 0.0033 0.02237 ± 12.6 >0.0001 0.8 tricaprylin CY + 31 ± 3.8 0.0008 0.012 38 ±6.8  >0.0001 0.7 tricaprin

Example 21 Chemoprotection Studies

The effect of nonanoic acid and lauric acid on in vivo induction ofimmune cell proliferation or protection assessed by the protocoldescribed in example 16. Significant increases in proliferation orprotection of bone marrow and spleen cell counts were observed with thepre-treatment of lauric acid in CY-treated mice. However, nonanoic aciddemonstrates weak (not significant) activity on immune cell counts(table 7) compared to cyclophosphamide treated mice.

TABLE 7 Effect of cyclophosphamide (CY), CY + nonanoic acid and CY +lauric acid on bone marrow and spleen cells Bone Marrow Spleen # CellsP/ # Cells P/ (×10⁶) Control P/CY (×10⁶) Control P/CY Control  58 ± 11.8 99 ± 22 CY 32 ± 6.3 0.0016 1.0 24 ± 6 0.0002 CY + 36 ± 5.6 0.0044 0.2628 ± 4 0.0004 0.27 nonanoic acid (6.25 μMole) CY + 42 ± 7.8 0.0185 0.0432 ± 5 0.0005 0.03 lauric acid (6.25 μMole)

Example 22 Chemoprotection Studies

The effect of trilaurin and trimyristin on in vivo induction of immunecell proliferation or protection assessed by the protocol described inexample 16. Trilaurin and trimyristin have weak (not significant)activity on bone marrow and spleen cell counts on CY-immunosuppressedmice (table 8).

TABLE 8 Effect of cyclophosphamide (CY), CY + trilaurin and CY +trimyristin on bone marrow and spleen cells Bone Marrow Spleen # CellsP/ # Cells P/ (×10⁶) Control P/CY (×10⁶) Control P/CY Control 49 ± 7.3105 ± 23   CY 27 ± 2.8 0.0014 19 ± 6.5 0.0007 CY + 31 ± 6.8 0.0028 0.219 28 ± 19.5 0.0004 0.302 trilaurin (6.25 μM) CY + 31 ± 9.9 0.0067 0.40215 ± 4.6 0.0007 0.314 trimyristin (6.25 μM)

Example 23 Chemoprotection Studies

The effect of tricaproin and sodium caproate on in vivo induction ofimmune cell proliferation or protection assessed by the protocoldescribed in example 16. Tricaproin and sodium caproate have a weak (notsignificant) activity on bone marrow and spleen cell counts onCY-immunosuppressed mice (table 9).

TABLE 9 Effect of cyclophosphamide (CY), CY + tricaproin and CY + sodiumcaproate on bone marrow and spleen cells Bone Marrow Spleen # Cells P/ #Cells P/ (×10⁶) Control P/CY (×10⁶) Control P/CY Control 48 ± 4.9 98 ±24.2 CY 25 ± 4.9 >0.0001 33 ± 13.2 0.0018 CY + 29 ± 4.1 0.0001 0.17 37 ±8.7  0.0035 0.51 tricaproin (6.25 μMole) CY +  39 ± 17.9 0.2403 0.09 35± 10.6 0.0026 0.77 sodium caproate (6.25 μMole)

Example 24 Chemoprotection Studies

The effect of sodium caprylate and sodium caprate on in vivo inductionof immune cell proliferation or protection assessed by the protocoldescribed in example 16.

A significant increase in proliferation or protection of bone marrowcell count was observed with pre-treatment with sodium caprylate andsodium caprate in CY-treated mice (FIG. 9).

Example 25 Chemoprotection Studies: Post-Treatment Regimens

Chemoprotection studies were performed as described in example 16 exceptthat mice were (post)-treated with MCT, sodium caprylate, sodium caprateor capric acid per o.s. on day 1, 2, 3 and 4.

A significant increase in bone marrow cell count was observed with thepost-treatment of MCT, sodium caprylate and sodium caprate in CY-treatedmice (table 10). When used as post-treatment, capric acid induces asignificant increase in spleen cell count and a weak increase in bonemarrow cell count (table 11).

TABLE 10 Effect of cyclophosphamlde (CY), CY + MCT, CY + sodiumcaprylate and CY + sodium caprate post-treatmeat on bone marrow andspleen cells Bone Marrow Spleen # Cells # Cells (×10⁶) P P/CY (×10⁶) PP/CY Control 52 ± 6.17 110 ± 29.3 CY 19 ± 4.99 >0.0001 30 ± 9.5 0.0007CY + 26 ± 3.70 >0.0001 0.0189 38 ± 7.2 0.0014 0.163 MCT (12.5 μM) CY +26 ± 5.33 >0.0001 0.0455  36 ± 12.5 0.0009 0.394 sodium caprylate (12.5μM) CY + 29 ± 4.45 0.0001 0.0140 28 ± 6.3 0.0007 0.696 sodium caprate(12.5 μM)

TABLE 11 Effect of cyclophosphamlde (CY), CY + capric acidpost-treatment on bone marrow and spleen cells Bone Marrow Spleen #Cells P/ # Cells P/ (×10⁶) Control P/CY (×10⁶) Control P/CY Control 48 ±7.9  88 ± 15.9 CY 31 ± 6.7 0.0026 21 ± 4.3 0.0001 CY + 37 ± 7.8 0.03260.209 31 ± 8.7 0.0001 0.035 capric acid (3.125 μMole) CY + 38 ± 4.60.0274 0.066 25 ± 6.3 0.0001 0.187 capric acid (6.25 μMole) CY + 38 ±7.4 0.0412 0.134 36 ± 7.8 0.0002 0.003 capric acid (12.5 μMole)

Example 26 Chemoprotection Studies: Immunophenotyping Assay

Female, 6- to 8-week old, C57BL/6 mice were pre-treated on day -3, -2and -1 per o.s. or intravenously at day 0 with different concentrationsof MCT. Immunophenotyping was also performed on immunosuppressedanimals. Immununosuppression was achieved with 80 mg/kg of5-fluorouracil (5-FU) or 100 to 200 mg/kg of cyclophosphamide (CY) or 12mg/kg of taxotere (TX) injected i.v. on day 0. Mice were sacrificed onday 5 by cardiac puncture. Blood and spleens were collected and cellsuspensions prepared and erythrocytes lysed in ACK buffer (155 mM NH₄Cl,12 mM NaHCO₃, 0.1 mM EDTA, pH 7.3) for 5 minutes. The cells were washedthree times in PBS, pH 7.4 and resuspended in tissue culture medium. Thecells were then incubated for 45 minutes on ice with fluoresceinisothiocyanate (FITC) or phycoerythrin (PE) conjugated cell surfacemarker according to the manufacturer's (Gibco/BRL, Cedarlane, BoehringerMannheim) recommendation. The cells were then washed in PBS, fixed with1% paraformaldehyde and analyzed with a Coulter XL flow cytometer.Analysis of the cell subsets was undertaken by determination of standardcell surface markers which were as follows: TCR (T-cell receptor), CD4(T helper), CD8 (T cytotoxic/suppressor), CD11b (macrophage), NK (NKcells) and Ly5 (B-cells). Bone marrow cells were obtained as describedin example 15. Cells were stained by a 45 minutes incubation of FITC orPE conjugated cell surface marker according to the manufacturer'srecommendation. The cells were then washed in PBS, fixed with 1%paraformaldehyde and analyzed with a Coulter XL flow cytometer. Analysisof the cell subsets was undertaken by determination of standard cellsurface markers which were as follows: CD34 (hematopoietic progenitorcells), CD41 (platelets, megakaryocytes), CD13 (myelomonocytic stemcells, myelocytes, promonocytes) and CD38 (lymphoid stem cells, pro-B,pre-B). Table 12 represents the effect of MCT on blood and spleenimmunophenotyping in normal mice. On blood immunophenotyping, MCTincrease CD8+ and LY5+ cell subsets. In some experiments, MCT increasesweakly the LY5-TCR-subset (data not shown). On spleen immunophenotyping,MCT increases significantly the relative percentage of LY5+TCR+ and CD4+cells. LY5-TCR- are non B-non T-cells that may represent theneutrophils.

When administered to immunosuppressed mice, MCT increases the relativepercentage of LY5-TCR- (probably neutrophils) and CD11+ (macrophage)cells on blood and spleen immunophenotyping compared to cyclophosphamidealone. These cell subsets origmate from the myeloid cell precursor(table 13).

TABLE 12 Effect of MCT on blood and spleen immunophenotyping in normalmice Cell subsets Control 6.25 μM 12.5 μM 50 μM Blood ImmunophenotypingCD8+ 12.76 ± 1.23 16.41 ± 1.16 — 13.18 ± 2.08 p ≦ 0.0004 p = 0.68 LY5+15.57 ± 6.91  24.0 ± 4.92 — 26.75 ± 4.11 p < 0.037 p < 0.01 SpleenImmunophenotyping LY5−TCR− 13.02 ± 2.54 — 16.84 ± 0.83 — p < 0.0257 CD4+ 19.9 ± 1.09 22.25 ± 1.64 —   22 ± 0.47 p ≦ 0.013 p < 0.091

TABLE 13 Effect of MCT on blood and spleen immunophenotyping incyclophosphamide (CY, 200 mg/kg) immunosuppressed mice Cell subsets CY6.25 μM 12.5 μM 50 μM Blood Immunophenotyping LY5− 36.82 ± 9.93 — 51.67± 11.10 46.32 ± 5.63 TCR− p ≦ 0.05 p = 0.1254 CD11+ 26.41 ± 4.54 — 42.12± 8.77 42.56 ± 8.62 p < 0.0119 p < 0.0098 Spleen Immunophenotyping LY5− 20.2 ± 4.05 23.92 ± 1.61 — — TCR− p < 0.07 (weak) CD11+ 16.31 ± 4.8527.47 ± 11.48 — — p ≦ 0.06 (weak)

Example 27 Chemoprotection Studies: Immunophenotyping Assay

Immunophenotyping of trimyristin, trilaurin, capric acid and sodiumcaproate was undertaken following the protocol described in example 26.

Table 14 represents the effect of these MCT analogues on blood andspleen immunophenotyping. On blood, trimyristin and trilaurin have nosignificant effect compared to cyclophosphamide alone. However onspleen, trimyristin and trilaurin enhance the relative percentage ofCD11+. Furthermore, trilaurin induces a significant increase in theLY5-TCR- and NK+ cell subsets.

Interestingly, capric acid and sodium caproate significantly increasethe relative percentage of LY5-TCR- on blood. On spleen, capric acid hasno significant effect compared to cyclophosphamide alone.

TABLE 14 Effect of trimyristin, trilaurin, capric acid and sodiumcaproate on blood and spleen immunophenotyping in cyclophosphamide (CY,200 mg/kg) immunosuppressed mice Compounds Cell subsets CY 6.25 μM 12.5μM Blood Immunophenotyping Trimyristin No significant effect TrilaurinNo significant effect Capric acid LY5−TCR− 56.36 ± 7.26 61.52 ± 5.1670.79 ± 3.95 p = 0.189 p < 0.0029 Sodium LY5−TCR− 40.91 ± 8.84 52.43 ±10.16 — caproate p = 0.063 (weak) Spleen Immunophenotyping TrimyristinCD11+ 16.31 ± 4.85 42.94 ± 8.45 — p ≦ 0.0002 Trilaurin CD11+ 16.31 ±4.85 43.94 ± 4.78 — p < 0.0001 LY5−TCR− 73.17 ± 1.41 77.86 ± 2.94 — p <0.0097 NK+  7.53 ± 2.52 17.46 ± 5.80 — p < 0.0067 Capric acid Nosignificant effect Sodium Not performed caproate

Example 28 Chemoproteetion Studies: Bone Marrow Immunophenotyping

The effect of MCT, sodium caprylate, sodium caprate on bone marrowimmunophenotyping was assessed by the protocol described in example 26.Treatment with cyclophosphamide induces a significant increase in allstudied subsets (CD34+, CD13+, CD41+ and CD38+). Addition of MCT orsodium caprylate or sodium caprate amplifies the number of CD13+ lineagewhich are myelomonocytic stem cells, myelocytes and promonocytes. Thisincrease in the relative percentage of CD13+ is significant compared tocyclophosphamide alone. The results clearly demonstrate that MCT andother related compounds induce a significant increase in the number ofbone marrow cells (as exemplified in the previous examples) and furtherenhance the relative percentage of precursor of phagocytic cells (PMNand monocytes). This may result in a better recovery from cytotoxictreatment or protection against infectious agents (table 15).

TABLE 15 Effect of MCT, sodium caprylate and sodium caprate on bonemarrow immunophenotyping in cyclophosphamide (CY, 200 mg/kg)immunosuppressed mice % Cells CD34+ CD13+ CD41+ CD38+ Control  1.1 ± 0.30.8 ± 0.2 1.6 ± 0.2 29.8 ± 6.5 Cyclophosphamide   10 ± 1.0 3.2 ± 0.5 4.2± 0.6 39.6 ± 13.6 (CY) CY + MCT 11.2 ± 1.3 4.5 ± 0.5 4.5 ± 0.4   42 ±15.7 p < 0.001 CY + Sodium 11.2 ± 1.3 4.9 ± 1.2 4.6 ± 1.3   36 ± 9.7caprylate p < 0.017 CY + Sodium caprate  9.1 ± 3.1 4.7 ± 1.7 3.7 ± 0.744.3 ± 22.8 p < 0.06

Example 29 Chemoprotection Studies

The effect of tridecanoyl serinol and didecanoyl serinol on in vivoinduction of immune cell proliferation or protection was assessed by theprotocol described in example 16.

As shown in table 16, tridecanoyl serinol significantly enhances thespleen cell counts. No significant effect was demonstrated on bonemarrow cell counts.

TABLE 16 Effect of cyclophosphamide (CY), CY + tridecanoyl serinol andCY + didecanoyl serinol on bone marrow and spleen cells Bone MarrowSpleen # Cells P/ # Cells P/ (×10⁶) Control P/CY (×10⁶) Control P/CYControl 53 ± 4.8 113 ± 15.5 CY 28 ± 3.4 >0.0001 29 ± 9.2 >0.0001 CY + 28± 4.6 >0.0001 0.95 42 ± 8.4 >0.0001 0.035 tridecanoyl serinol CY + 30 ±3.8 >0.0001 0.54 36 ± 9.9 >0.0001 0.27 didecanoyl serinol

Example 30 Chemoprotection Studies

The effect of α-methyl tridecanoyl-L-fucopyranose and β-methyltridecanoyl-L-fucopyranose on in vivo induction of immune cellproliferation or protection was assessed by the protocol described inexample 16.

As shown in table 17, β-methyl tridecanoyl-L-fucopyranose demonstratedweak (not significant) activity on bone marrow cell counts compared tocyclophosphamide treated mice. The lack of activity of the α-methylanomer is expected in view of the known instability of α-alkylpyranosides.

TABLE 17 Effect of cyclophosphamide (CY), CY + α-methyl tridecanoyl-L-fucopyranose and CY + β-methyl tridecanoyl-L-fucopyranose on bonemarrow Bone Marrow # Cells P/ (×10⁶) Control P/CY Control   53 ± 8.0 CY26.2 ± 2.6 0.0058 CY + α-methyl tridecanoyl-L-fucopyranose 30.4 ± 9.30.0133 0.334 CY + β-methyl tridecanoyl-L-fucopyranose 34.6 ± 8.5 0.00680.061

Example 31 Chemoprotection Studies

The effect of ethyl caprate and capric acid N,N-dimethylacetamide esteron in vivo induction of immune cell proliferation or protection wasassessed by the protocol described in example 16.

As shown in table 18, only capric acid N,N-dimethylacetamidesignificantly enhances the bone marrow cell count. No significant effectwas demonstrated on spleen cell counts.

TABLE 18 Effect of cyclophosphamide (CY), CY + ethyl caprate and CY +capric acid N,N-dimethylacetamide on bone marrow Bone Marrow # Cells(×10⁶) P/Control P/CY Control 50.2 ± 4.8 CY 27.5 ± 8.0 0.0031 CY + ethylcaprate 27.5 ± 4.4 0.0032 1.0 CY + capric acid N,N-dimethyl-acetamide37.4 ± 5.9 0.042 0.036

Example 32 Antitumor Activity

Female 6-8-week old C57BL/6 mice were injected intravenously on day 0with 1×10⁵ B16F10 melanoma cells from ATCC (source of cell culture, Dr.I. J. Fidler). Animals were then injected i.v. with or without MCT (25μMole/mouse) on day 7, 9, 14 and 16 and 10 mg/kg Doxorubicin on day 10and 17. Mice were sacrificed on day 22. Body weight and tumor volumewere recorded. Serial tumor volume was obtained by bi-dimensionaldiameter measurements with calipers, using the formula 0.4 (a×b²) where“a” was the major tumor diameter and “b” the minor perpendiculardiameter.

This experiment was conducted to verify if MCT is not exacerbating orprotecting the cancer cells rather than immune cells.

FIG. 10 represents the chemoprotective effect and anti-tumor efficacy ofMCT in combination with a sub-therapeutic concentration of doxorubicinin B16F10 melanoma model. MCT induces a weak reduction (T/C around 20%)of the tumor volume as close as the sub-therapeutic concentration ofdoxorubicin (TIC around 25% reduction) when used alone. An additiveeffect is observed when MCT is used in combination with doxorubicin (T/Caround 45 to 50%). These results indicate that it is possible to attaintherapeutic activity when MCT is combined with a sub-therapeuticconcentration of cytotoxic drugs.

Example 33 Antitumor Activity

The syngeneic tumor DMBA3 (DA-3, breast carcinoma model) arose from apreneoplastic lesion treated with 7,12-dimethylbenzanthracene in femaleBALB/c mice. DA-3 cells were grown as monolayer cultures in plasticflasks in RPMI-1640 containing 0.1 mM nonessential amino acids, 0.1 μMsodium pyruvate, 2 mM L-glutamine and 100 μg/ml gentamycin sulfate. Thiswas further supplemented with 50 μM 2-mercaptoethanol and 10% fetalbovine serum. The DA-3 tumors were serially passage in vivo by s.c.inoculation of 5×10⁵ viable tumor cells to produce localized tumors in6- to 8-week old BALB/c mice. The animals were then serially monitoredby manual palpation for evidence of tumor. Serial tumor volume wasobtained by bi-dimensional diameter measurements with calipers, usingthe formula OA (a×b²) where “a” was the major tumor diameter and “b” theminor perpendicular diameter. Tumors were palpable, in general, 7-10days post-inoculation.

Two treatment regimens were used for anti-tumor efficacy and protectionevaluation of MCT in combination with cyclophosphamide (CY, 100 mg/kg)and taxotere (TX, 20 mg/kg) in the DA-3 tumor model. BABL/c mice wereinjected with tumor cells on day 0. Treatment with MCT was done per oson day 6, 7 and 8; day 13, 14 and 15; day 20, 21 and 23 followed bytreatment with CY or TX administered i.v. as single bolus injection onday 9 and 16. Body weights and tumor volumes were monitored from day 4until day 23. At day 23, all animals were sacrificed. The % T/C(treatment over control) was calculated as the ratio of tumor volumes attermination date in the treatment group divided by the respectivevolumes in the control group multiplied by 100. By the NCl criteria theproduct is considered effective if % T/C is ≦40%.

This experiment was performed to verify if MCT is not exacerbating orprotecting the cancer cells rather than immune cells. FIG. 11 shows thechemoprotective effect and anti-tumor efficacy of MCT in combinationwith sub-therapeutic concentration of CY and TX in DA-3 breast carcinomamodel. MCT induces a weak reduction (T/C around 18%) of the tumor volumecompared to the control. When MCT is used in combination with CY or TX,no exacerbation of the tumor volume is observed. However, when used incombination with CY, a therapeutic response is observed (T/C=39.4%).These results indicate that therapeutic activity may be attained whenMCT is combined with a sub-therapeutic concentration of CY. This effectmay be due to an overall increase in immune cell efficiency inMCT-treated animals (FIG. 11 and table 19).

TABLE 19 Effect of MCT on tumor volume in combination with asub-therapeutic concentration of cyclophosphamide (CY, 100 mg/kg) andtaxotere (TX, 20 mg/kg) Tumor volume Treated/Control (%) Control 58.8 ±60.1 CY 27.5 ± 15.9 46.8 TX 37.9 ± 41.5 64.5 CY + MCT 23.2 ± 13.1 39.4TX + MCT 38.8 ± 31.0 66.1 MCT 48.5 ± 35.2 82.5

Example 34 Antitumor Activity

Antitumor and chemoprotection efficacy were assessed by the protocoldescribed in example 30, with the exception of use of a therapeuticconcentration of cytotoxic drugs (cyclophosphamide, 200 mg/kg; taxotere,30 mg/kg).

This experiment was conducted to verify if MCT is not exacerbating orprotecting the cancer cells rather than immune cells. FIG. 12 shows thechemoprotective effect and anti-tumor efficacy of MCT in combinationwith therapeutic concentration of CY and TX in DA-3 breast carcinomamodel. MCT induces a weak reduction of the tumor volume compared to thecontrol. When MCT is used in combination with CY or TX, no exacerbationof the tumor volume is observed. When treated with CY or CY+MCT, asignificant reduction of the tumor volume is observed. Furthermore, asignificant response in reduction of tumor volume is attained with thetreatment of MCT combined to TX (p<0.0327) compared to TX alone which isnot significant compared to the control mice (p=0.1211) (table 20).These results indicate that a therapeutic activity may be attained whenMCT is combined with a non-significant therapeutic concentration of TX.This effect may be due to an overall increase in immune cell efficiencyin MCT-treated animals.

TABLE 20 Effect of MCT on tumor volume in combination with a therapeuticconcentration of cyclophosphamide (CY, 200 mg/kg) and taxotere (TX, 30mg/kg) Treatment T/C (%) P/Control P/CY P/TX Control MCT 0.4299 CY 18.80.0337 CY − MCT 22.1 0.0022 0.2928 TX 64.7 0.1211 TX − MCT 46.7 0.03270.5468

All references cited in this document are hereby incorporated byreference herein in their entireties.

Modifications and variations of the compositions and methods describedherein will be obvious to those skilled in the art from the foregoingdescription. Such modifications and variations are intended to comewithin the scope of the appended claims.

We claim:
 1. A method of stimulating hematopoiesis in a patient in needof such stimulation, said method comprising administering to the patienta pharmacologically effective amount of a composition comprising one ormore compounds described by formula I

wherein Y=O or NH; R₁ is a C₇ or C₉ alkyl; and A and B are independentlyhydrogen or R₁C(O); and wherein said patient undergoes bone marrowtransplantation or has a condition selected from neutropenia ormyelosuppression arising from chemotherapy or radiotherapy.
 2. Themethod, according to claim 1, wherein the condition is myelosuppressionarising from chemotherapy.
 3. The method, according to claim 1, whereinthe condition is myelosuppression arising from radiotherapy.
 4. Themethod, according to claim 1, wherein the condition is chronicneutropenia.
 5. The method, according to claim 1, wherein the conditionis transient neutropenia.
 6. The method, according to claim 1, whereinthe condition is neutropenia arising from a hematologic disease.
 7. Themethod, according to claim 1, wherein the condition is drug-inducedneutropenia.
 8. The method, according to claim 1, wherein the conditionis neutropenia arising from a nutritional deficiency.
 9. The method,according to claim 1, wherein the condition is neutropenia arising frominfection.
 10. The method, according to claim 1, wherein the conditionis neutropenia arising from radiotherapy.
 11. The method, according toclaim 1, for the treatment of a patient undergoing bone marrowtransplantation.
 12. The method, according to claim 1, wherein thecompound of formula I is caprylic acid triglyceride.
 13. The method,according to claim 1, wherein the compound of formula I is capric acidtriglyceride.
 14. The method, according to claim 1, which comprisesadministering to the patient a mixture of two compounds, wherein A and Bare R₁C(O) and R₁ is C₇ and C₉ alkyl, respectively.
 15. The method,according to claim 14, wherein R₁ is CH₃(CH₂)₆— and CH₃(CH₂)₈—,respectively.
 16. The method, according to claim 1, which comprisesadministering four geometric isomers of C₈ and C₁₀ fatty acidtriglycerides described by the following formula:


17. The method, according to claim 1, wherein the patient is undergoingseparate or simultaneous administration of a human colony-stimulatingfactor.
 18. The method, according to claim 17, wherein thecolony-stimulating factor is Granulocyte colony-stimulating factor(G-CSF) or Granulocyte macrophage colony-stimulating factor (GM-CSF).19. The method, according to claim 1, wherein the patient is undergoingsimultaneous administration of a human cytokine.
 20. The method,according to claim 19, wherein the cytokine is interleukin 2 orinterleukin 15.